The proposed project will reveal synaptic circuit abnormalities caused by methyl-CpG-binding protein 2 (MeCP2) deficiencies in cortical pyramidal neurons. The overall goal of this project is to identify cortical circuit deficits associated with deleted and knocked down levels of MeCP2 in order to reveal how this gene influences synaptic circuit organization. It is already known that mutations in MeCP2 are responsible for the disease Rett syndrome, which presents with severe motor and cognitive symptoms, and that abnormalities in MeCP2 expression are also present within the autism spectrum. However, it is not known whether MeCP2 influences cortical circuit organization, or whether its effects are specific to any cortical region. Thus, the proposed study will analyze synaptic circuits of four populations of pyramidal cells in the mouse cortex, including those in cortical layers 2/3 and 5 in both somatosensory and motor cortices. For each population of cells, synaptic inputs to neurons deficient in MeCP2 will be compared to those of normal cells. The effects of MeCP2 abnormalities will be assessed through the use of two model systems. First, MeCP2-null mice will be compared to wild type littermate controls in order to reveal the circuit phenotype of neural populations entirely lacking in MeCP2. Second, mice that have been electroporated in utero with plasmids encoding small interference RNAs to silence MeCP2 expression will have the gene knocked down in a subpopulation of cortical cells. By comparing the circuit phenotype of knockdown cells with neighboring wild type cells, the cell-specific effects of MeCP2 on circuit organization will be isolated from its network- wide effects. Circuit phenotypes will be assayed using laser scanning photostimulation (LSPS), a highly sensitive and efficient electrophysiologically based imaging tool for mapping synaptic connectivity. LSPS combines whole-cell patch clamp recording from a post-synaptic neuron while focally uncaging glutamate in the surrounding tissue via flash photolysis. When performed on slice preparations of neocortex, LSPS reveals patterns of monosynaptic input onto individual cells. By using this technique it will be possible to elucidate circuitry abnormalities associated with deletion or knockdown of MeCP2 in cortex. The proposed project will contribute significantly to the understanding of how MeCP2 influences cortical circuit organization. By doing so, the results of this study are expected to have direct implications for understanding the critical 'wiring'defects in the cortex of the brain in Rett syndrome.